Topical agents containing vitamin D3 (VD3) analogues such as calcipotriol, maxacalcitol and tacalcitol and the combination of calcipotriol/betamethasone dipropionate (betamethasone) are prescribed for patients with psoriasis. However, they are known to occasionally cause hypercalcemia, and the frequency of hypercalcemia is suggested to vary according to the VD3 analogue used. In this study, to address the reason for these differences, the calcemic effects of maxacalcitol-, calcipotriol- and calcipotriol/betamethasone-containing ointments in rats were evaluated. The serum calcium levels in rats treated with ointments containing maxacalcitol, but not calcipotriol or calcipotriol/betamethasone, were significantly elevated, which is consistent with clinical observations. The serum concentration of VD3 analogue in rats treated with ointments containing calcipotriol and calcipotriol/betamethasone was lower than that in rats treated with maxacalcitol-containing ointment. Thus, the calcemic effects appear to be associated with the systemic exposure of VD3 analogues in rats. To understand the mechanism underlying the different systemic exposures of VD3 analogues, skin permeation and metabolic stability of VD3 analogues were evaluated. The cumulative amount of calcipotriol permeated through rat skin was significantly lower than that of maxacalcitol. On the other hand, the metabolic clearance of calcipotriol in rat hepatocytes was higher than that of maxacalcitol. Similar results were obtained using human skin and human hepatocytes. The current study demonstrates that the lower calcemic effects of calcipotriol- and calcipotriol/betamethasone-containing ointments are caused by the low systemic exposure of calcipotriol according to low skin permeability and rapid hepatic elimination after topical application.